DESCRIPTION The project aims at deciphering the mechanism of intramolecular transfer of protons in cytochrome c oxidase (COX). Being a terminal member of the mitochondrial and bacterial respiratory chain, COX is a key enzyme of aerobic respiration and energy transduction in most eukaryotes and many bacteria. The enzyme reduces molecular oxygen to water and utilizes the free energy of this strongly exergonic reaction to drive the electrogenic translocation of protons across the membrane. The mechanism of intramolecular transfer of electrons and protons in COX and of the coupling between them has been a central research problem in bioenergetics for more than 50 years. Very recently, the high resolution structure of the enzyme has been solved by X-ray diffraction, which allows for focused investigation into the molecular mechanism of proton pumping by the enzyme. There are two apparent channel-like domains connecting the oxygen- reducing heme-copper binuclear center with the negatively charged aqueous phase, and these channels have been suggested to be involved in redox-linked uptake and conduction of protons. There are several highly conserved protonatable amino acid residues within these channels, most notably K362, E286 and D132 in subunit I, that have been shown to be absolutely necessary for intramolecular proton transfer. Replacements of these residues by site-directed mutagenesis strongly reduces enzyme steady state catalytic activity. In this project, the investigators wish to resolve partial steps of intramolecular proton transfer in COX from R. sphaeroides with the aid of time-resolved measurements of membrane potential generation developed in Moscow and then to take site- specific mutant forms of the enzyme available from the Urbana laboratory and determine which individual steps of proton pumping are affected by the mutations in the specific proton channel residues. They hope these experiments will elucidate the specific roles of the two proton channels in the reaction mechanism of COX and the molecular mechanism of proton translocation within the channels.